Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications is found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.
Bacterial infections are a serious problem in humans. In the past decade, the number of “supergerms” that resist treatment has increased dramatically. Unfortunately, the very same arsenal of drugs used to overcome these microbes helped give rise to antibiotic-resistant strains of bacteria. Of great importance are several antibiotic-resistant and sometimes fatal bacteria including S. aureus, Pseudomonas aeruginosa (pneumonia), and Enterococcus faecalis (urinary tract and blood infections).
For most healthy people, these antibiotic-resistant bacteria are not life-threatening. The immune system, the body's natural defense against microbes, usually fights off disease-causing bacteria. However, when bacteria attack people with weakened immune systems they can be deadly. Hailed as miracle drugs, antibiotics have cured thousands of bacterial infections, from acne to strep throat to ear infections. Today, there are more than 100 types of antibiotics on the U.S. market. Due to increasing resistance to antibiotics, however, new treatments are still needed.
In particular, S. aureus infections have been problematic to treat. S. aureus are non-mobile, non-sporulating gram-positive cocci 0.5–1.5 μm in diameter, that occur singly and in pairs, short chains, and irregular three-dimensional grape-like clusters. S. aureus can grow over a wide range of environmental conditions, but they grow best at temperatures between 30° C. and 37° C. and at a neutral pH. They are resistant to desiccation and to chemical disinfection, and they tolerate NaCl concentrations up to 12%. It has been found that the growth of S. aureus becomes unusually sensitive to high NaCl concentrations (by decreasing Ca2+ concentration) in growth media allowing for autolysis (29).
The global regulatory locus agr encodes a two-component, quorum sensing system that is involved in the generation of two divergent transcripts, RNAII and RNAIII, from two distinct promoters, P2 and P3, respectively. RNAIII is the regulatory molecule of the agr response, hence responsible for the up-regulation of extracellular protein production and down-regulation of cell-wall associated protein synthesis during the postexponential phase (39,49). The RNAII molecule, driven by the P2 promoter, encodes a four-gene operon, agrBDCA, with AgrC and AgrA corresponding to the sensor and activator proteins of a two component regulatory system. Additionally, agrD, in concert with agrB, participates in the generation of an octapeptide with quorum sensing functions (31,41). The autoinducing peptide would stimulate the transcription of the agr regulatory molecule RNAIII which ultimately interacts with target genes to modulate transcription (49) and possibly translation (44).
In contrast to agr, the sarA locus activates the synthesis of both extracellular (e.g. α- and β-hemolysins) and cell-wall proteins (e.g. fibronectin binding protein) in S. aureus (15). The sarA locus is composed of three overlapping transcripts [sarA P1 (0.56 kb), sarA P3 (0.8 kb) and sarA P2 (1.2 kb) transcripts], each with a common 3′ end but initiated from three distinct promoters (P1, P3 and P2 promoters). Due to their overlapping nature, each of these transcripts encodes the major 372-bp sarA gene, yielding the 14.5 kDa sarA protein (2). DNA footprinting studies have shown that the sarA protein binds to the promoters of several target genes (19) including agr, hla (alpha hemolysin gene), spa (protein A gene) and fnbA (fibronectin binding protein A gene), thus implicating sarA as a regulatory molecule that can modulate target gene transcription via both agr-dependent and agr-independent pathways (9,19,20). With agr-dependent pathway of target gene activation, the sarA protein binds to the agr promoter to stimulate RNAIII transcription and RNAIII, in turn, interacts with target genes (e.g. hla) to modulate transcription. With sarA-dependent but agr-independent pathway, the SarA protein will interact directly with target-gene promoters (e.g. hla and spa) (19) to control gene transcription. Deletion and promoter fusion analyses indicates that the regions upstream of the sarA P2 and between the P1 and P3 promoters have a modulating role in sarA expression, possibly by controlling transcription from the sarA P1 promoter, the predominant promoter within the sarA locus (10,39) (FIG. 1A).
A great need exists for methods and compositions which can affect or regulate the virulence of bacteria, such as the expression of sarA and the resultant virulence determinants of S. aureus and other bacteria.